CN106635926B

CN106635926B - Bacillus safensis YJC-4 and application thereof in prevention and treatment of tobacco black shank and growth promotion

Info

Publication number: CN106635926B
Application number: CN201710060678.2A
Authority: CN
Inventors: 陈德鑫; 林北森; 夏长剑; 李锡宏; 刁朝强; 徐同伟; 简胜义; 杨艺炜; 文锦涛; 张晓强; 黄翔; 聂忠扬
Original assignee: Hainan Co Of China National Tobacco Corp; Tobacco Research Institute of CAAS; Guiyang Office of Guizhou Tobacco Corp; Tobacco Research Institute of Hubei Province
Current assignee: Hainan Co Of China Tobacco General Co; Tobacco Research Institute of CAAS; Guiyang Office of Guizhou Tobacco Corp; Tobacco Research Institute of Hubei Province
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2020-01-21
Anticipated expiration: 2037-01-25
Also published as: CN106635926A

Abstract

A strain of Bacillus safensis YJC-4 is classified as Bacillus safensis, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2016, 4 and 15 days, the address is No.3 of Xilu No.1 of Beijing Kogyo, and the preservation number is CGMCC No. 12356. The strain has the following advantages: (1) the biocontrol effect is good: in a pot experiment, the YJC-4 bacterial liquid is used for treating a tobacco plant inoculated with the tobacco black shank bacterium, the control effect reaches 81.59 percent, is obviously superior to the control effect of 58 percent of methylfrost-manganese-zinc and a biological preparation of bacillus subtilis, has potential application value in the aspect of biological control of the tobacco black shank, and has good development and application prospects. The biological control is environment-friendly, is not easy to generate drug resistance, is safe to people and livestock, and creates conditions for the green prevention and control development of the tobacco. (2) The growth promoting effect is good: the biomass and the agronomic characters of the YJC-4 bacterial liquid tobacco strain have obvious promotion effects and are obviously different from those of a control group.

Description

Bacillus safensis YJC-4 and application thereof in prevention and treatment of tobacco black shank and growth promotion

Technical Field

The invention belongs to the technical field of biological control, and particularly relates to bacillus safensis YJC-4 and application thereof in controlling tobacco black shank and promoting growth.

Background

Tobacco Black Shank (Phytophthora parasitica var. nicotianae) is a facultative parasitic bacterium, can induce Tobacco Black Shank (tobaco Black Shank), and has destructive damage to Tobacco. The phytophthora parasitica prefers high temperature and high humidity, when the temperature is 28-30 ℃ and the humidity reaches more than 80%, favorable conditions are created for the release of sporangium of the phytophthora parasitica and the propagation of zoospores, and the prevalence and the occurrence of tobacco phytophthora parasitica are most easily caused at the moment, so that the tobacco plants die in a large area. Tobacco is used as a host plant, the disease reaches a peak stage from the agglomeration stage to the vigorous growth stage, and the root and stem base of the tobacco plant are mainly damaged. In addition, the continuous cropping of the land for many years, the mixed occurrence of the continuous cropping, the tobacco bacterial wilt and the root knot nematode disease aggravate the harm to the health of tobacco plants, and result in the reduction of the yield of tobacco leaves and the reduction of the quality of the tobacco leaves.

The soil mainly contains 3 microorganisms of bacteria, fungi and actinomycetes, and researches show that a plurality of bacilli as antagonistic bacteria have good prevention and treatment effects on the tobacco black shank. A plurality of researches at home and abroad show that the Bacillus subtilis, the Bacillus amyloliquefaciens, the Bacillus pumilus and the like have good control effects on the tobacco black shank through action mechanisms of antibiotic, bacteriolysis, competition, heavy parasitism and the like. Some antagonistic bacteria have the growth promoting effect on tobacco plants, promote the absorption of nitrogen, phosphorus and potassium of tobacco seedlings, and can effectively reduce the morbidity and disease index of diseased tobacco seedlings.

At present, commercial bacillus subtilis exists, but the bacillus subtilis still has defects in the aspects of tobacco black shank prevention and growth promotion.

Disclosure of Invention

Aiming at the problems, the invention aims to provide a bacillus safensis strain which can effectively prevent and control the black shank of tobacco and can obviously promote the growth of the tobacco.

A strain of Bacillus safensis YJC-4 is classified as Bacillus safensis, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2016, 4 and 15 days, the address is No.3 of Xilu No.1 of Beijing Kogyo, and the preservation number is CGMCC No. 12356.

Furthermore, the thallus of the bacillus safensis YJC-4 is rod-shaped, and gram staining shows positive.

Further, the components of the culture medium of the bacillus safensis YJC-4 are as follows: 3.0g of beef extract, 5.0g of peptone, 1.0g of yeast powder, 10.0g of glucose, 18.0g of agar and 1L of distilled water.

Further, the culture conditions of the bacillus safensis YJC-4 are as follows: shaking and culturing at constant temperature of 30-35 deg.C and pH7-8 for 24-72 h. Preferably, the culture is performed for 60 hours at 35 ℃ and pH7.5 with constant temperature shaking.

The application of the bacillus safensis YJC-4 in preventing and controlling tobacco black shank is provided.

Further, the application form of the bacillus safensis YJC-4 in the prevention and treatment of the tobacco black shank is bacterial liquid, the application form is root irrigation, and the root irrigation amount of each tobacco seedling is 1 multiplied by 10⁷-9×10⁷cfu。

Furthermore, the root irrigation is carried out in a form of irrigating the roots for three times, wherein the root irrigation is carried out once every 7 days after the tobacco plants grow to 5-6 true leaves for the first time. Furthermore, the root irrigation amount is the same in three times.

The application of the bacillus safensis YJC-4 in promoting the growth of tobacco.

Further, the application form of the bacillus safensis YJC-4 in promoting the growth of tobacco is bacterial liquid, the application mode is root irrigation, and the root irrigation amount of each tobacco seedling is 1 multiplied by 10⁷-9×10⁷cfu。

Further, in order to make the antagonistic bacteria better colonize the root of the tobacco plant, the tobacco seedling is planted at 1X 10 before being transplanted into the growing field⁶-9×10⁶And (3) soaking roots in cfu/mL YJC-4 bacterial liquid for 20-40 min.

The invention separates and screens 1 strain of bacillus fomentarius YJC-4 which has better antagonistic activity to tobacco black shank bacteria and good growth promoting effect to tobacco strains from tobacco rhizosphere soil, and has the following advantages:

(1) the biocontrol effect is good: in a pot experiment, the YJC-4 bacterial liquid is used for treating a tobacco plant inoculated with the tobacco black shank bacterium, the control effect reaches 81.59 percent, is obviously superior to the control effect of 58 percent of methylfrost-manganese-zinc and a biological preparation of bacillus subtilis, has potential application value in the aspect of biological control of the tobacco black shank, and has good development and application prospects. The biological control is environment-friendly, is not easy to generate drug resistance, is safe to people and livestock, and creates conditions for the green prevention and control development of the tobacco.

(2) The growth promoting effect is good: the YJC-4 bacterial liquid has obvious promotion effects on the biomass and the agronomic characters of tobacco plants, and has obvious difference with a control group.

Drawings

FIG. 1 shows the inhibitory effect of antagonistic bacterium YJC-4 on tobacco phytophthora parasitica (A is a control, and B is a YJC-4 strain);

FIG. 2 is the morphological structure of the antagonist bacteria YJC-4 under a scanning electron microscope;

FIG. 3 influence of temperature on the growth of YJC-4 strain;

FIG. 4 influence of pH on the growth of YJC-4 strain;

FIG. 5 Effect of culture time on the growth amount of YJC-4 strain;

FIG. 6 16S rDNA amplification band of YJC-4;

FIG. 7 phylogenetic tree of the 16S rDNA sequence of antagonist strain YJC-4;

FIG. 8 growth of several differently treated tobacco plants.

Bacillus safensis (Bacillus safensis) YJC-4 is preserved in China general microbiological culture Collection center (CGMCC), the address is No.3 Siro No.1 of Beijing market and the area facing the sun, the preservation date is as follows: 2016, 4 months and 15 days, and the preservation number is CGMCC No. 12356.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings.

The materials used in all examples are as follows:

the main reagents and materials were as follows:

the DNA rapid extraction Kit Easy Pure Bacteria Genomic DNA Kit is a product of the whole gold biotechnology limited company (the cargo number EE 161-01);

the gram staining kit is a solibao product (cat # G1060);

the 58 percent wettable powder of the first frost and the manganese zinc is produced by Jiangsu Lingbao chemical company Limited;

the bacillus subtilis wettable powder is produced by Deqiang biology company;

other medicaments are all Chinese medicament analytically pure.

Tobacco variety: honghua Dajinyuan (susceptible variety) provided by the national tobacco improvement center.

Phytophthora nicotianae (Phytophthora nicotianae): provided by the national tobacco improvement center.

The culture medium used for the isolation, culture and determination of the antagonistic bacteria is as follows:

the antagonistic bacteria are cultured and separated by using nutrient agar medium NA (beef extract 3.0g, peptone 5.0g, yeast powder 1.0g, glucose 10.0g, agar 18.0g, distilled water 1L, pH6.8 adjusted) and nutrient broth medium NB (beef extract 3.0g, peptone 5.0g, yeast powder 1.0g, glucose 10.0g, distilled water 1L).

The determination of antibacterial activity adopts oat culture medium OA (18 g of oatmeal, 18.0g of agar, 1L of distilled water, and pH adjusted to 6.8).

The physiological and biochemical characteristics are measured by using a sports culture medium (gelatin 80g, beef extract 3g, peptone 10g, sodium chloride 5g, agar 4g, distilled water 1L, pH 6.8).

Citrate agar medium (MgSO)₄·7H₂O 0.2g，NH₄H₂PO₄1g，K₂HPO₄2g of sodium citrate, 80mg of bromothymol blue, 15g of agar and 1L of distilled water, and the pH value is adjusted to 6.8).

For anaerobic growth, glucose broth (8 g nutrient broth, 50mL of 20% glucose stock solution, 950mL of distilled water, adjusted to pH7.0) was used.

Example 1 screening of Bacillus safensis YJC-4 and its antagonistic action against Heiguobacter nigricans

1.1, separation and screening of antagonistic bacteria:

collecting 87 parts of soil from Guizhou Kaiyang, Qingzhen, Sichuan Panzhihua, Wenchang, Shandong Zhucheng, Yishui, Jimo and the like, selecting the soil at the root of the black shank plant and the soil at the root of the adjacent healthy plant in the depth of 5-10cm for obtaining the high-efficiency antagonistic bacteria, marking, and storing in a refrigerator at the temperature of-20 ℃ for later use.

The 87 collected soils were treated as follows: firstly, removing impurities such as roots, stems, leaves and the like in soil, crushing and sieving by a 20-mesh sieve. Weighing 5g of treated soil by using an electronic balance, putting the soil into a 100mL conical flask, adding water to a constant volume of 50mL, uniformly shaking, and taking 1mL of supernatant to perform gradient dilution to 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6And (4) doubling. By pipette guns respectivelyAnd sucking 200 mu L of each diluted solution, uniformly coating the diluted solution on the NA solid culture medium by using a coater respectively, sealing the culture medium, and inversely placing the culture medium in an incubator at 37 ℃ for constant temperature culture for 48 hours. Sterile water was used as a blank control, 3 replicates. And (3) selecting 324 single colonies with different growth characteristics as candidate bacteria of the antagonistic bacteria, and performing purification culture (selecting the single colonies by using an inoculating loop, streaking on an NA solid culture medium, sealing, placing in an incubator at 37 ℃ for constant temperature culture for 48h, and repeating the steps for 1-2 times to obtain purified single colonies).

1.2 antagonistic action of antagonistic bacteria candidate on phytophthora parasitica:

inoculating tobacco black shank bacteria to the center of an OA plate culture medium by using a sterilized puncher (the diameter is 10mm) by using a confronting culture method, drawing two short lines on two parallel sides of the tobacco black shank bacteria block at a position 25mm away from the center (after a small amount of bacteria liquid is dipped by an inoculating loop, drawing two lines on the culture medium, and the length is about 0.5cm), wherein the treating group is obtained, and a blank OA culture solution without candidate bacteria is adopted as a control group. Then the mixture is inversely placed in a constant temperature incubator at 28 ℃ for 4 days, whether inhibition bands exist around the black shank is observed, and the inhibition diameter d (mm) of the mixture is measured by a cross method. Controls were not inoculated with candidate bacteria, all else being identical.

The formula for calculating the bacteriostasis rate is as follows:

the bacteriostatic ratio (%) × (control group colony diameter-treatment group colony diameter)/treatment group colony diameter ] × 100%.

Of 324 candidate bacteria, 12 bacteria have the bacteriostatic diameter of 39.90-58.20mm, and the relative inhibition rate is 46-73%, as shown in Table 1. The YJC-4 strain has the best bacteriostatic effect on the tobacco black shank, the bacteriostatic diameter of the strain reaches 58.20mm, the relative inhibition rate reaches 72.30 percent, and the strain has a better bacteriostatic effect as shown in figure 1.

TABLE 1 plate antagonistic Effect of antagonistic bacteria on tobacco Black shank

Note that the difference of the lower case English letters after the number is significant (P < 0.05), and the same applies below.

1.3 morphological identification of Strain YJC-4

On the NA culture medium, YJC-4 bacterial colonies are all milky white, smooth in surface and opaque.

The morphology and the structure of the YJC-4 strain are observed by an electron scanning microscope, and the steps are as follows: (1) sampling: inoculating the YJC-4 strain into an NB culture medium, carrying out shake constant-temperature culture for 48h, taking a bacterial liquid sample, centrifuging (3000rpm), removing a supernatant, adding 0.01mol/L PBS buffer solution with the pH value of 7.0, and fully washing. (2) Fixing: immobilization was performed with 2.5% glutaraldehyde and 2% osmic acid and washed with PBS. (3) And (3) dehydration treatment: performing sample treatment by using PBS (phosphate buffer solution) of ethanol according to concentration gradient of 30%, 50%, 70% and 90%, fully dehydrating by using 100% ethanol, dehydrating by using a mixed solution of ethanol and tert-butyl alcohol for 10min, and finally placing in tert-butyl alcohol for dehydration treatment. (4) And (5) freeze drying. (5) After spraying the Pt powder, observing the morphological structure of the Pt powder on an electron scanning microscope. FIG. 2 shows that the strain YJC-4 has a rod-shaped thallus, endogenetic spores with a size of 0.2-0.4X 1.0-2.0. mu.m.

1.4 identification of physiological and biochemical traits

Reference is made to "method for studying plant diseases" (edited by Ching-shoda, published by agricultural Press of China), and the gram-positive/negative stain, motility, oxidase, anaerobic growth in glucose medium, hydrolysis of starch, citrate utilization, V.P. test, growth of 7% sodium chloride, and the like are identified for strain YJC-4.

The results show that YJC-4 strain shows purple color when observed in a microscope, is a gram-positive bacterium, has certain motility, shows negative oxidase and shows positive V.P. test, can utilize citrate, cannot hydrolyze starch, cannot grow under anaerobic conditions in a glucose culture medium and grows in 7 percent sodium chloride.

1.5 optimal culture conditions for Strain YJC-4

1.5.1 Effect of temperature on growth of Strain YJC-4: inoculating YJC-4 strain into a liquid NB culture medium with the pH value of 7.0 by the inoculation amount of 5 percent, uniformly shaking, respectively placing the strain in a constant-temperature incubator with the temperature of 20 ℃, 25 ℃, 27.5 ℃, 30 ℃, 32.5 ℃, 35 ℃, 40 ℃ and 45 ℃ for culturing for 2d at 120r/min, and measuring the concentration of antagonistic bacteria by an ultraviolet spectrophotometer to reflect the growth amount of the strain.

As shown in fig. 3: with the increase of the temperature, the growth amount of the YJC-4 antagonistic bacteria generally tends to be rapidly increased and then rapidly reduced, and the optimal temperature is 35 ℃ and then is 32.5 ℃. The growth is severely inhibited under the conditions of high temperature and low temperature.

1.5.2 Effect of pH on growth of Strain YJC-4: inoculating YJC-4 strain into a liquid NB culture medium by an inoculation amount of 5%, wherein the initial pH values are 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 respectively, shaking uniformly, fermenting for 2d at 120r/min in an incubator at 30 ℃, and determining the concentration of antagonistic bacteria.

As shown in fig. 4: with the increase of the pH, the growth amount of the YJC-4 antagonistic bacteria generally shows a tendency of 'rapidly increasing and then rapidly decreasing', the optimum pH is 7.5, and then is 7.0, and when the pH is less than 5.5, bacterial colonies hardly grow; when the pH value is more than 9.0, the colony growth amount is reduced seriously, which indicates that the YJC-4 antagonistic bacteria cannot resist strong acid and strong alkali. The difference of pH values between treatments has a significant difference on the growth amount of the YJC-4 antagonistic bacteria, which indicates that the pH value is an important factor for the growth of the YJC-4 antagonistic bacteria.

1.5.3 Effect of fermentation time on growth of Strain YJC-4: inoculating the YJC-4 strain into a liquid NB culture medium by an inoculation amount of 5%, carrying out shake culture in an incubator at 30 ℃ at 120r/min, and sampling for antagonistic bacteria concentrations of 0h, 12h, 24h, 36h, 48h, 60h and 72h respectively.

As shown in fig. 5: with the increase of the culture time, the growth amount of the YJC-4 antagonistic bacteria generally shows a trend of 'rapidly increasing and then basically keeping unchanged', the growth amount is the highest in 60 hours, and the growth amount is 48 hours secondly.

1.6 identification of 16S rDNA of Strain YJC-4

(1) According to the requirements and steps of the DNA rapid extraction kit, 1mL of YJC-4 bacterial solution cultured overnight is taken, cells are cracked through reuspen Buffer11 and lysine Buffer, and impurities are removed through RNase A, Binding Buffer11, Clean Buffer and Wash BufferFinally, eluting by an Elution Buffer to obtain the genomic DNA of the strain YJC-4, and storing at-20 ℃. (2) Amplifying a 16SrDNA sequence by using the genomic DNA of the strain YJC-4 as a template and 799F (5'-AACAGGATTAGATACCCTG-3') and R1492(5'-GGTTACCTTGTTACGACTT-3') as primers; 50 μ L amplification system: template 1. mu.L, forward and reverse primers 1. mu.L each, 2 × Easy

25 mu L of PCR Super Mix and 22 mu L of ultrapure water; PCR reaction procedure: 94 ℃ for 5min, 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 1min, 35 cycles, 72 ℃ for 10 min. The PCR product was electrophoresed through 1.5% TAE agarose gel and EB stained to give a bright band, a 16SrDNA band of YJC-4 on the right side, which shows a molecular size of 750bp, as shown in FIG. 6. (3) The PCR product entrusts Qingdao optisco biotechnology limited to carry out sequence determination, the length of the 16S rDNA nucleotide sequence of the bacterium is determined to be 670bp, the sequence information is shown in SEQ ID No.3 in a sequence table, and the GenBank accession number is KX 663830; (4) sequencing results homology comparisons were performed using BLAST software, and phylogenetic trees were constructed using the Neighbor-Joining method of MEGA 5.1. As can be seen from FIG. 7, YJC-4 has a recent evolutionary relationship with Bacillus safensis (JF411315.1) and has 99% homology. According to the plate bacteriostasis effect, morphological identification, physiological and biochemical character analysis, optimum growth condition exploration and 16S rDNA sequence homology analysis, the YJC-4 is concluded to be identified as Bacillus safensis (Bacillus safensis).

Example 2 Pot culture test of Bacillus safensis YJC-4 for controlling tobacco black shank

Three safflower big-golden tobacco plants which grow to 5-6 true leaves are taken to be potted and inoculated with black shank bacteria under the same conditions: the root base of the tobacco plant is slightly scratched by a knife, and the cultured black shank is inoculated to the wound.

FIG. 8 shows the growth of three different treated tobacco plants: no.1 is that after the phytophthora parasitica is inoculated, 20mL (2 +/-0.5) multiplied by 10 of roots are irrigated immediately⁶Growth condition of cfu/mL YJC-4 antagonistic bacterial liquid after 7 days; no. 2 shows the disease condition of tobacco plants without using YJC-4 antagonistic bacteria 3 days after the inoculation of the phytophthora parasitica; no.3 shows that Y is not used 7 days after the black shank bacterium inoculationJC-4 antagonistic bacteria and the disease state of tobacco plants. The antagonistic bacterium liquid has a good prevention and treatment effect on the black shank, and is beneficial to further research.

Example 3 Pot culture test of Bacillus safensis YJC-4 control Effect on tobacco Black shank control

The concentration of the shake-cultured YJC-4 bacterial liquid is about (2 +/-0.5) x 10⁸cfu/mL, diluting by 100 times, carrying out root irrigation treatment on the safflower Honghuadajinyuan tobacco plants growing to 5-6 true leaves in a pot plant, and simultaneously inoculating the tobacco plants with black shank bacteria under the same conditions: the root base of the tobacco plant is slightly scratched by a knife, and the cultured black shank is inoculated to the wound. The dose was administered every 7 days for 3 times. A control medicament is prepared by respectively spraying 500 times of 58% methylosine-manganese-zinc wettable powder and 1000 hundred million/g bacillus subtilis powder to inoculate tobacco strains of the phytophthora parasitica, a blank control is treated by the same amount of sterile water, the rest is the same, and the specific operation is shown in Table 2. Each treatment was 20, 3 replicates. The incidence (%) and disease index and relative control effect (%) were investigated 7 days after the third application treatment. The grading standards were carried out according to the tobacco industry standards of the people's republic of China (YC/T39-1996).

The incidence rate is the number of diseased plants/total number of tobacco plants multiplied by 100%.

The disease index is 100 × Σ (number of disease plants at each stage × representative value at each stage)/(total number of investigated plants × representative value at highest stage).

The plants were examined in grades.

Level 0: the whole plant is disease-free.

Level 1: stem lesions do not exceed 1/3 for stem girth, or leaf wilting below 1/3.

And 3, level: the stem scabs surround the stem girth 1/3-1/2, or 1/3-1/2 leaves are slightly withered, or the lower few leaves have scabs.

And 5, stage: the stalk lesion exceeds 1/2 of the stalk circumference, but does not completely surround the stalk circumference, or 1/2-2/3 leaves wither.

And 7, stage: the stem lesions all encircle the stem circumference, or more than 2/3 leaves wither.

And 9, stage: the diseased plants die basically.

Relative prevention and treatment effect (%) (disease index of control group-disease index of treatment group)/disease index of control group x 100%.

The data are subjected to variance analysis and calculation by adopting a Duncan new complex polarization method of a DPS data processing system.

Table 2 medicament test concrete operation table

The potted plant test result in the table 3 shows that the YJC-4 antagonistic bacteria have better prevention and treatment effects 7 days after the third drug application treatment, the morbidity of the tobacco plants treated by the YJC-4 antagonistic bacteria is 11.13 percent, the disease index is 5.32, and the prevention and treatment effects reach 81.59 percent; compared with 58% of cream-methyl and manganese-zinc, the incidence rate of the strain treated by YJC-4 is slightly higher, but the difference is not obvious and is on the same level; the disease index of tobacco plants treated by YJC-4 is minimum, the control effect is highest, and the control effect is not obvious from the difference between the tobacco plants treated by 58 percent of cream-methyl-manganese-zinc and 1000 hundred million/g of bacillus subtilis. The result shows that the effect of the YJC-4 antagonistic bacteria on the black shank is the best, and is equivalent to the effect of 58 percent of methylzinc-manganese wettable powder and 1000 hundred million/g of bacillus subtilis, and the YJC-4 antagonistic bacteria have better popularization value.

TABLE 3 greenhouse control effect of strain YJC-4 on tobacco black shank

Labeling: data represent mean ± sem, data were obtained using Duncan's new repolarization method, with different lower case and upper case letters indicating significant differences at P <0.05 and P <0.01 levels, respectively.

Example 4 growth promotion test for Bacillus shafu YJC-4

To verify the growth promoting effect of YJC-4 antagonistic bacteria on tobacco plants, firstly, tobacco seedlings growing to 5-6 true leaves are removed from the temporary planting tray and put into a prepared antagonistic bacteria liquid (1X 10) to be tested⁶cfu/mL), taking out after 30min, and transplanting in a flowerpot filled with sterilized soil. After transplanting and seedling revival for 3 days, respectively irrigating the tobacco plants with antagonistic bacteria liquid, 10 mL/plant,each replicate was 10, 3 replicates and the blank was treated with an equal amount of clear water. The culture was observed under greenhouse conditions at a temperature of 28 ℃ and a humidity of about 80%. Respectively selecting 10 tobacco plants from 30 tobacco plants at 15d, 30d and 45d after transplanting, recording growth promotion indexes, measuring fresh mass, dry mass, fresh weight of roots and dry weight of the roots of the whole tobacco plants, and analyzing the growth promotion effect of the antagonistic bacteria on the biomass of the tobacco plants; measuring the average plant height, the average root length, the maximum leaf length and the maximum leaf width, and analyzing the promotion effect of the antagonistic bacteria on the agronomic characters of the tobacco plants.

Growth promoting effect of YJC-4 antagonistic bacteria on biomass of tobacco plants:

as shown in table 4, the growth promoting effect of the antagonistic bacteria on the biomass was investigated as the tobacco plants grow, and the influence of the antagonistic bacteria on the fresh quality, the dry quality, the fresh weight and the dry weight of the whole tobacco plants was investigated 15, 30 and 45 days after transplantation. Research shows that the antagonistic bacteria has obvious promotion effect on the biomass of the tobacco plant.

Investigation is carried out on 24 days (15 days after transplanting) in 9 months, the YJC-4 antagonistic bacteria can promote the growth of the biomass of tobacco plants, wherein the promotion effect on the dry weight of roots is most obvious, the YJC-4 is very different from CK, and the growth rate reaches 38.46 percent. The difference of the growth promoting effect of other indexes is not obvious, which shows that the promoting effect of the antagonistic bacteria on the biomass of the tobacco plants is not obvious 15 days after the transplantation.

According to investigation on 09 days after 10 months (30 days after transplanting), compared with a control, the accelerating effects of the YJC-4 antagonistic bacteria on the fresh quality, the dry quality, the fresh weight and the dry weight of the whole tobacco plant are obvious and all reach very significant differences. The accelerating effect of the YJC-4 antagonistic bacteria on the dry weight of the root is obvious, and the growth rate is 38.46 percent.

According to investigation of 10 months and 24 days (45 days after transplanting), compared with a control, the YJC-4 antagonistic bacteria have obvious promotion effects on the fresh mass of the whole tobacco plant, the dry mass of the whole tobacco plant, the fresh weight of roots and the dry weight of the roots, and all the promotion effects reach very significant differences, the YJC-4 antagonistic bacteria have the most obvious promotion effects on the dry mass of the whole tobacco plant and the dry weight of the roots, the growth rates are respectively 50.00% and 62.09%, the promotion effect of the antagonistic bacteria on the biomass of the tobacco plant is obvious, and the material accumulation of the tobacco plant can be effectively promoted.

TABLE 4 growth promoting effect of antagonistic bacteria on tobacco plant biomass

Note that the upper case letters of the same column data indicate significant difference (P < 0.05), the upper case letters indicate significant difference (P < 0.01), and the following is the same.

The growth promoting effect of YJC-4 antagonistic bacteria on the agronomic characters of tobacco plants is as follows:

as shown in Table 5, the growth promoting effect of antagonistic bacteria on the agronomic traits was investigated, and the influence of antagonistic bacteria on plant height, root length, maximum leaf length, and maximum leaf width was investigated 15, 30, and 45 days after transplantation, respectively. Research shows that the antagonistic bacteria has obvious promotion effect on the agronomic characters of the tobacco plants.

Investigation is carried out for 24 days (15 days after transplanting) in 9 months, and the YJC-4 antagonistic bacteria can obviously promote the agronomic characters of tobacco plants, wherein the promotion effect on the root length, the maximum leaf length and the maximum leaf width is most obvious, the difference is extremely obvious compared with CK, and the difference of the plant height is obvious compared with CK.

Investigation is carried out on 09 days (30 days after transplanting) within 10 months, and the promoting effects of the YJC-4 antagonistic bacteria on the plant height, the root length, the maximum leaf length and the maximum leaf width of the tobacco plant are obvious and all reach very significant differences, wherein the promoting effects of the YJC-4 antagonistic bacteria on the plant height and the maximum leaf length are most obvious, the growth rate on the plant height is 17.03%, and the growth rate on the maximum leaf length is 17.88%.

Investigation is carried out for 24 days (45 days after transplanting) in 10 months, the promotion effects of the YJC-4 antagonistic bacteria on the plant height, the root length, the maximum leaf length and the maximum leaf width of the tobacco plant are obvious and all reach extremely significant differences, the promotion effect of the YJC-4 antagonistic bacteria on the root length is obvious, and the growth rate is 25.59 percent. The result shows that the YJC-4 antagonistic bacteria have obvious growth promoting effect on the biological properties of tobacco plants.

TABLE 5 growth promoting action of antagonistic bacteria on agronomic traits of tobacco plants

SEQUENCE LISTING

<110> Hubei province tobacco science research institute; tobacco institute of the Chinese academy of agricultural sciences; guiyang city company of tobacco company of Guizhou province

<120> Bacillus saxatilis YJC-4 and application thereof in prevention and treatment of tobacco black shank and growth promotion

<130>2017

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<170>PatentIn version 3.5

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cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 180

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aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 360

accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 420

gtgctacaat ggacagaaca aagggctgca agaccgcaag gtttagccaa tcccataaat 480

ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 540

tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 600

ccacgagagt ttgcaacacc cgaagtcggt gaggtaacct ttatggaccc cccccccaaa 660

gggggggcaa 670

Claims

1. A strain of Bacillus safensis YJC-4 is classified as Bacillus safensis and is characterized in that the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2016, 4 and 15 days, the address is No.3 of No.1 Siro of North Chen West Lu in the Inward region of Beijing, and the preservation number is CGMCC No. 12356.

2. The use of Bacillus safensis YJC-4 of claim 1 in the prevention and treatment of tobacco black shank and the promotion of tobacco growth.

3. The use of claim 2, wherein the application is in the form of bacterial liquid, the application is in the form of root irrigation, and the root irrigation amount of each tobacco seedling is 1 x 10⁷-9×10⁷cfu。

4. The use as claimed in claim 3, wherein the root watering is carried out in three portions, the first time when the tobacco plant has grown to 5-6 true leaves, and the subsequent time every 7 days.

5. The use of claim 4, wherein the three root crops are of the same amount.

6. Use according to claim 4, wherein the tobacco seedlings are at 1 x 10 before being transplanted into the growing field⁶-9×10⁶And (3) soaking roots in cfu/mL YJC-4 bacterial liquid for 20-40 min.